1. Field of the Invention
The present invention relates to analysis of fluorescence resonance energy transfer.
2. Related Background Art
Fluorescence resonance energy transfer (FRET) is a phenomenon in which excitation energy transfers from a fluorescent molecule to another molecule. The molecule that supplies energy to another molecule is called a donor, and the molecule that receives the energy is called an acceptor. When FRET occurs, the fluorescence of the donor weakens. When the acceptor is a fluorescent molecule, fluorescence is emitted from the acceptor.
When FRET in a cell is measured by microscopic observation, the following method may be used: measure the fluorescence intensities of the donor and the acceptor when the donor is excited, and calculate the ratio between the measured intensities, i.e., the acceptor's intensity/the donor's intensity (see Atsushi Miyawaki et al., “Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin,” Nature, vol. 388, pp. 882-887, 28 Aug., 1997). The fluorescence intensities of the donor and acceptor can be measured in turn by switching the bandpass filters disposed in front of the detector. Moreover, the fluorescence from the donor and the acceptor can be simultaneously detected with two detectors by separating the fluorescence from them with a dichroic mirror and then filtering the fluorescence with the bandpass filter. As the detector, a camera such as a cooled CCD camera, or a photomultiplier is used, for example.
By this method, change in the fluorescence where the fluorescence of the donor weakens and that of the acceptor intensifies, which is characteristic of the FRET, can be observed. Calculating the fluorescence intensity ratio between the donor and the acceptor clearly shows the amount of the change in the fluorescence intensities. Moreover, this method is advantageous because it can cancel variation in the fluorescence intensities due to the thickness of the cell, the distribution of the dyes and the illumination unevenness of the light source. However, this method is unfit for quantitative measurement of the fluorescence intensity ratio though it can detect the changes in the FRET. When the ratio between the amounts of the donors and the acceptors in a cell is changed, when the wavelength region where fluorescence is detected is changed, when the kind of the fluorescent reagent in use is changed, or when the spectral sensitivity characteristic of the detector is changed, the value of the fluorescence intensity ratio changes accordingly. Consequently, no quantitative comparison can be made between the measurement values obtained before and after these experimental conditions are changed.
An FRET efficiency (Et) is an example of a value that can be quantitatively compared even when the experimental condition changes. Et=1−Fd′/Fd is known as an expression for obtaining this, where Fd is the fluorescence intensity of the donor when no FRET occurs, and Fd′ is the fluorescence intensity of the donor when FRET occurs. To determine Fd, light with the absorption wavelength of the acceptor is used to illuminate a specimen containing the donor and acceptor, all the acceptor molecules in measurement region are broken by photo-bleaching, and then the fluorescence intensity of the donor is measured. By Measuring Fd′ at time intervals before this bleaching experiment, variations of Et over time can be calculated.
When the bleaching experiment is performed after the measurement of Fd′, the wavelength of the illumination light and the dichroic mirror are switched from ones for Fd′ measurement to ones for bleaching. Intense light not including the absorption wavelength region of the donor and including the absorption wavelength region of the acceptor as widely as possible is used to illuminate the specimen. This is done in order to bleach the acceptor as quickly as possible without the fluorescence of the donor being affected by bleaching or the like. However, the wavelength region of the light for bleaching frequently overlaps with the fluorescence wavelength region of the acceptor. Accordingly, part of the light for bleaching leaks from the dichroic mirror and enters the detector for monitoring the fluorescence of the acceptor. Since the leakage light has a very high intensity, it may be impossible to monitor the bleaching process of the acceptor by the detector.
For example, a case is assumed where the fluorescent dye ECFP is used as the donor, the fluorescent dye EYFP is used as the acceptor and the fluorescence of the donor and acceptor are measured after the separation with a filter. FIG. 7 shows the absorption spectrum 51 and the fluorescence spectrum 52 of ECFP and the absorption spectrum 53 and the fluorescence spectrum 54 of EYFP. Considering these spectra, the following bandpass filters are used: the filter having a transmission wavelength region “a” for exciting ECFP at 440 nm with the half width of 20 nm, the filter having a transmission wavelength region “c” for filtering the fluorescence of ECFP at 480 nm with the half width of 30 nm, and the filter having a transmission wavelength region “d” for filtering the fluorescence of EYFP at 535 nm with the half width of 25 nm. In this case, a dichroic mirror that allows light with wavelengths not less than 455 nm to pass therethrough is used. Thereafter, in bleaching EYFP, a bandpass filter that has a transmission wavelength region “b” at 525 nm with the half width of 45 nm is used. The filter is selected so as not to include the absorption wavelength region of ECFP and widely cover the absorption wavelength region of EYFP. In this case, a dichroic mirror of 560 nm is used (see Atsushi Miyawaki, “GFP wo mochiita bunshikan FRET (Intermolecular FRET Using GFP),” Seitai no Kagaku, Vol. 53, No. 1, pp. 75-81, Feb., 2002, and Asako Sawano et al., “Multicolor Imaging of Ca2+ and Protein Kinase C Signals Using Novel Epifluorescence Microscopy,” Biophysical Journal, Vol. 82, pp. 1076-1085, Feb., 2002). When an ND filter or the like is placed on the optical path, it is removed from the path in order to apply light as intense as possible to the specimen. Accordingly, part of this intense illumination light enters the detector for measuring EYFP in the EYFP bleaching experiment.
Therefore, in order to determine when the bleaching is completed and the illumination is to be stopped, the specimen is illuminated by the light for bleaching over a predetermined time, then the settings of the dichroic mirror, the filter and the like of the microscope are returned to the ones for the fluorescence measurement, and then the fluorescence of the acceptor is measured. Thereafter, the settings are returned to the ones for the bleaching, and the light for bleaching is used to illuminate the specimen. This operation is repeated until the fluorescence of the acceptor no longer attenuates (see Atsushi Miyawaki and Roger Y. Tsien, “Monitoring Protein Conformations and Interactions by Fluorescence Resonance Energy Transfer between Mutants of Green Fluorescent Protein,” Methods in Enzymology, vol. 327, pp. 472-500, 2000).
Furthermore, there is a method for measuring the bleaching speed of the fluorescence of the donor to quantitatively measure the FRET efficiency. In this method, the FRET efficiency Et is expressed as Et=1−τb1/τ′b1, where τb1 is the bleaching speed of the donor fluorescence when no FRET occurs, and τ′b1 is the bleaching speed of the donor fluorescence when FRET occurs. The bleaching speed of the donor fluorescence can be obtained as the attenuation speed of the fluorescence intensity when the fluorescence intensity is measured a number of times at time intervals while the specimen is continuously illuminated by the light with the absorption wavelength of the donor. However, since the fluorescence of the donor is bleached in the measurement, variations over time of the FRET efficiency for the same specimen cannot be determined. In addition, it is necessary to set a region for determining τb1 in the specimen and previously break the acceptor molecules in the region by photo bleaching (see Fred S. Wouters et al., “FRET microscopy demonstrates molecular association of non-specific lipid transfer protein (nsL-TP) with fatty acid oxidation enzymes in peroxisomes,” The EMBO Journal, Vol. 17, No. 24, pp. 7179-7189, 1998, and Philippe I. H. Bastiaens et al., “Imaging the molecular state of proteins in cells by fluorescence resonance energy transfer (FRET) Sequential photobleaching of Forster donor-acceptor pairs,” Proceedings of the Second Hamamatsu International Symposium on Biomolecular Mechanisms and Photonics: Cell-Cell Communication, pp. 77-82, 1995).